No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells

ABSTRACT Essential genus-specific genes have not been discovered for fowl adenovirus (FAdV), which hampers the development of FAdV-based vectors and attenuated FAdV vaccines. Reverse genetics approaches were employed to construct FAdV-4 mutants carrying deletions or frameshift mutations covering the whole left and right ends of the viral genome. The results of virus rescue and plaque forming experiments illustrated that all the 22 designated ORFs (open reading frames) were dispensable for the replication of FAdV-4 in chicken hepatoma Leghorn male hepatoma (LMH) cells and primary embryo hepatocytes. RNA-seq data demonstrated that ORF28 and ORF29 were not protein-encoding genes, and suggested a promoter (RP1) and an intron in these regions, respectively. The promoter activity of RP1 was further confirmed by reporter gene expression experiments. GAM-1-deleted FAdV-4 formed small plaques, while deletion of GAM-1 together with ORF22 resulted in even smaller ones in LMH cells. Simultaneous deletion of ORF28, ORF29, and GAM-1 led to growth defect of FAdV-4. These facts implied that genus-specific genes contributed to and synergistically affected viral replication, although no single one was essential. Notably, replication of FAdV-4 mutants could be different in vitro and in vivo. XGAM1-CX19A, a GAM-1-deleted FAdV-4 that replicated efficiently in LMH cells, did not kill chicken embryos because virus propagation took place at a very low level in vivo. This work laid a solid foundation for FAdV-4 vector construction as well as vaccine development, and would benefit viral gene function study. IMPORTANCE Identification of viral essential genes is important for adenoviral vector construction. Deletion of nonessential genes enlarges cloning capacity, deletion of essential genes makes a replication-defective vector, and expression of essential genes in trans generates a virus packaging cell line. However, the genus-specific essential genes in FAdV have not been identified. We constructed adenoviral plasmid carrying deletions covering all 22 genus-specific ORFs of FAdV-4, and found that all virus mutants could be rescued and amplified in chicken LMH cells except those that had defects in key promoter activity. These genus-specific genes affected virus growth, but no single one was indispensable. Dysfunction of several genus-specific genes at the same time could make FAdV-4 vectors replication-defective. In addition, the growth of FAdV-4 mutants could be different in LMH cells and in chicken embryos, suggesting the possibility of constructing attenuated FAdV-4 vaccines.

LMH cells. Deletion of ORF28, ORF29 and GAM-1 resulted in a defective FAdV-4. A GAM-1-deleted FAdV-4 that replicated efficiently in LMH cells did not kill chicken embryos in vivo. These results showed that no one specific gene was essential for FAdV-4 growth and that some genes may have overlapping functions that compensate for one another. Further, the results showed that the effects of specific mutations are context-specific depending on the cells used for assays. Finally, he authors used RNA-seq to annotate gene expression patterns in the genus-specific regions and used reporter assays to identify novel promoter regions in these areas. This is a comprehensive and convincing report that sheds significant new light on genus-specific genes of FAdV-4. The analyses are conducted in a controlled and technically correct manner. The data are validated using appropriate statistical analyses. The manuscript is very well written and accessible to a diverse audience. Overall, I find this study of general interest to the readership and I have no specific concerns that require revision.
Reviewer #2 (Comments for the Author): The authors introduced numerous deletions into the left and right end of FAdV genome and they conclude that not a single gene located on those genus specific areas is essential for growth in LMH cells. Altogether, the authors report "negative results" as they didn't found a single gene driving replication in LMH cells which seems a very ambitious hypothesis. Overall, the main outcome seems limited, it indicates that each gene be replaced as no clear adjunct with growth in either LMH or primary cells exists. Major concerns: Viruses deleted at the left end of genome carry a reported gene substituting ORF1-ORF-1B and ORF-2 which are obviously not needed for growth and plaque purification. However, from figure 1 it seems that the majority of recombinants contain larger parts being deleted not solely restricted to a single gene. It seems that rescued viruses were not passaged which does not allow to conclude on stability of the noticed phenotypic trait (lines 128-129 and 452-499). This is of special importance as obviously non-robust growth was seen in some cases (line 123). A more detailed description would be needed.
No scaling of the y-axis (µm) is given in figures. Fig 5: why was growth of M2829-CX19A already assessed at 3dpi in comparison to all others? Fig. 11 B: days after inoculation should be given, similar to 11A; why are numbers of eggs for which virus titer was determined different and not always 15? Lines 349: the explanation for the reduced growth of XGAM1-CX19A is not sound as embryos were infected at 6 days of incubation with nor or very limited impact of the immune system, not comparable with in ovo vaccination at day 18. Overall, this would have been a general feature for all mutants. In addition, growth of XGAM1-CX19A on LHM cells seems more rapid than XHE-CX198 (see figures 3 and 5).

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Response to the first reviewer expert:
The professor had no negative comments.
Response to the second reviewer expert:

Viruses deleted at the left end of genome carry a reported gene substituting
ORF1-ORF-1B and ORF-2 which are obviously not needed for growth and plaque purification. However, from figure 1 it seems that the majority of recombinants contain larger parts being deleted not solely restricted to a single gene.
What the reviewer professor pointed out is true. We explained this in the first subsection of "The strategy of constructing FAdV-4 mutants" in Results: The restriction site-defined regions of the viral genome were systematically deleted to detect the key regions for virus replication. Frameshift mutations or coding sequence (CDS) deletions were further introduced into the key regions to distinguish the effects of individual genes in the following steps.
After carefully considering the professor's comment, we changed the title to "No genus-specific gene is essential for the replication of fowl adenovirus 4 in chicken LMH cells". Such modification could make it more stringent. In the revised manuscript, we deleted the description of "single gene" in some places while reserve it in others where the term of "single genes" were used to contrast with combined several genus-specific genes.

It seems that rescued viruses were not passaged which does not allow to conclude on stability of the noticed phenotypic trait (lines 128-129 and 452-499).
This is of special importance as obviously non-robust growth was seen in some cases (line 123). A more detailed description would be needed.
For the first version of this manuscript, we only studied several FAdV-4 mutants with deletions at the right end. In that situation, all these mutants were purified. After that, we constructed more FAdV-4 mutants, and finally 13 of the total 18 rescued mutants were purified. Some FAdV-4 mutants were not purified because we thought purification was not necessary. For the unpurified viruses, only infectious titers were determined. All these rescued viruses were passaged at least 3 times on LMH cells to enrich sufficient progeny viruses for the following experiments. For these rescued viruses, plaques were formed and CPE occurred when linearized adenoviral plasmid was used to transfect LMH cells, suggesting an acceptable growth rate. When the seed viruses were amplified in LMH cells, the propagation of progeny viruses was For all purified or unpurified virus stocks, infectivity titer was determined on LMH cells by the limiting dilution assay in which mCherry+ or GFP+ cells were counted 30 hpi.

No scaling of the y-axis (µm) is given in figures.
For the cell images, the scale bars are identical for both x-and y-axes. We think it could be appropriate to label one scale bar for the whole figure. occurred. We thought that the rapid growth of M2829-CX19A could be illustrated more clearly by providing the cell image at days 2 and 3 post transfection. Fig. 11 B: days after inoculation should be given, similar to 11A; why are numbers of eggs for which virus titer was determined different and not always

15?
We did the modification as the professor suggested. To titrate virus from so many livers is very laborious. For these who survived the observation endpoint, livers from 6 embryos were randomly selected for virus titration because such number of samples could provide enough data for statistical analysis.
6. Lines 349: the explanation for the reduced growth of XGAM1-CX19A is not sound as embryos were infected at 6 days of incubation with nor or very limited impact of the immune system, not comparable with in ovo vaccination at day 18.
Overall, this would have been a general feature for all mutants. In addition, growth of XGAM1-CX19A on LHM cells seems more rapid than XHE-CX198

(see figures 3 and 5).
We agree with the professor. Although cells for innate immune system can be detected in 4-day-old chicken embryos [pubmed:31063007], there is no evidence that they can protect the embryos from virus infection. We searched and read some publications. However, we could not find pertinent knowledges to describe the difference of virus growth in cultured cells and in early chicken embryos. We choose to delete this sentence.
XHE-CX19A and XGAM1-CX19A seemed to have similar growth rate in LMH cells. Both propagated slower than FAdV4-GFP although the difference between XHE-CX19A and FAdV4-GFP was not statistically significant ( Figure 4 and Figure   6). In contrast, in primary chicken embryo hepatocytes, XHE-CX19A and FAdV4-GFP had similar growth rate while the propagation of XGAM1-CX19A was much slower (Figure 7). The results of virus growth in primary hepatocytes were consistent with the data from chicken embryo inoculation experiments. We re-wrote this paragraph as following: GAM-1-deleted FAdV-4 (XGAM1-CX19A) could grow in LMH cells and primary chicken hepatocytes, but the viability was already compromised, especially in primary cells. Its amplification in chicken embryos was very inefficient (Fig 11), suggesting that FAdV-4 was less dependent on GAM-1 for replication in vitro than in vivo. The mechanism behind this deserves further study. 7. Line 62: a more actual version of the reference should be used: Benkö et al.

(J.Gen:Virol.)
We updated the taxonomy of adenoviridae and cited the latest publication in the revised version. Thanks.

Line 79: Griffin et al. 2021 should be referenced
We cited this publication in the revised manuscript.

Line 285: viruses from livers of embryos
We corrected this mistake as the professor suggested. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey.
As an open-access publication, Spectrum receives no financial support from paid subscriptions and depends on authors' prompt payment of publication fees as soon as their articles are accepted. You will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.